CUEDC2and ATF6α Are Involved In Apoptosis Modulation In The Heart Under ER Stress

Background and Objective:The death of cardiac myocytes is a leading cause of acute heart failure and theprogression of chronic heart dysfunction. It has great clinical significance to study thedetailed molecular mechanisms of the death of cardiac myocytes and find new target toprevent cell death. In the heart, The endoplasmic reticulum (ER) is a multifunctionalorganelle that controls the synthesis, folding, assembly, and transport of proteins and alsoprovides a dynamic intracellular Ca2+storage compartment. Under conditions that perturbprotein folding in the ER, such as ER Ca2+depletion or energy/nutrients deprivations, theexpression of ER chaperones is induced through the unfolded protein response (UPR). Theacute UPR activation is thus an important adaptive response to ER stress. Increasedexpression of chaperones augments the protein folding and processing capacity and restoresequilibrium to the ER lumen. However, chronic ER stress leads to sustained UPR activation,and failure to suppress the proapoptotic aspect of the UPR often leads to ER stress relatedcell death. While the mechanisms that allow cells to escape apoptosis and adapt are not wellunderstood, this "decision" appears to require, at a minimum, the improvement of ERprotein folding and processing, which serves to attenuate further UPR signaling even as astressful stimulus persists. Also, how the three limbs of the UPR manage adaptation tochronic stress is not understood.CUEDC2(CUE domain containing protein2) is a newly identified protein. Thefunction of CUEDC2was not fully investigated. Previouly study confirmed that CUEDC2could interact with GADD34and recruit protein phosphotase1(PP1), leading to thedeactivation of IKKβ and modulation the activity of NF-κB. Beside this, CUEDC2canmodulate ER-α protein stability through the ubiquitin-proteasome pathway, also acts as acrucial determinant of resistance to endocrine therapies in breast cancer. Interestedly,CUEDC2is a key regulator of mitosis progression, and CUEDC2dysregulation maycontribute to tumour development by causing chromosomal instability. However, the role of CUEDC2in the heart is unknown. CUEDC2is highly expressed in the heart, indicating itmight play an important role under physiological and kinds of stress conditons. So, one ofour aims is to study the role of CUEDC2under ER stress in the heart.Hypoxic states are generally associated with cardiovascular disease. Adaptation tochronic hypoxia is one well-defined means of improving cardiac tolerance to certain kindsof stresses. However, the detailed mechanisms underlying myocardial adaptation to chronichypoxia are still poorly understood. Hypoxia activates unfolded protein response. However,the behavior of individual signaling pathways can vary markedly over time. The exactsignaling pathway under chronic hypoxia is unknown. We plan to identify the activation ofUPR and the function of it under chronic hypoxia.Methods:Part Ⅰ We firstly investigated the expression, interactionship and post-transcriptionalmodulation of CUEDC2using bioinformational methods, predicting the possible functionof CUEDC2in the heart. Then, we investigated the role of CUEDC2in the development,structure and physiological function of heart by using CUEDC2gene knockout mice andlittermate wildtype mice.Part Ⅱ To study the role and mechanism of CUEDC2under acute stress in the heart,we established an mouse model of ischemia reperfusion injury in vivo by ligating andreleasing the left anterior descending coronary artery, to evalute the role of CUEDC2inmyocardial infarction in CUEDC2gene knockout mice. Additionally, we isolated andcultured primary neonatal rat ventricular cardiomyocytes, knocked down the expression ofCUEDC2with adenovirus and studied the role of CUEDC2in cardiomyocytes duringischemia reperfusion injury.Part Ⅲ We constructed heart hypertrophy and heart dysfunction mouse model byarotic constriction, compared the differences in the degree of hypertrophy and heartfunction grade between the wild type and Cuedc2^-/-mice, and studied the molecularmechanisms of CUEDC2under chronic ER stress and in the progression of heartdysfunction.Part IV Samples taken from the right ventricular outflow tract were collected frompatients with cyanotic or acyanotic congenital heart disease. Protein level of CUEDC2wasevaluated by western blot. For the activity of UPR, the expression of Bip was examined byimmunohistochemistry, while the expression of XBP-1s, phorspho-eIF2, Bip, ATF6α and phorspho-PERK were tested by western blot. Then, primary rat cardiomyocytes werecultured and exposed to1.0%O2,5.0%CO2for different durations to establish the chronichypoxic cell model. After different duration of hypoxic exposure, cells were collected andsubjected to real time-PCR and western blot to detecte the mRNA and protein expression ofthe UPR.Results:1. CUEDC2is highly expressed in the heart. The growth and development of micecould not be affected by knockout of CUEDC2gene. Under physiological conditions, therewas no significant difference in heart size and cardiac function between the wild-type miceand Cuedc2^-/-mice.2. In vivo and in vitro experiment showed ischemia and reperfusion injury couldreduce CUEDC2protein expression significantly; the myocardial infarct size in theCuedc2^-/-mouse group after ischemia-reperfusion injury was reduced by about30%compared to the wild-type group; in the Cuedc2^-/-mouse myocardial tissue and the isolatedand cultured primary MEF cells, JNK activation could be notably inhibited by ischemia-reperfusion injury. In the primary cardiomyocytes, knockdown the expression of CUEDC2gene significantly inhibited JNK activation caused by ischemia-reperfusion injury, andincreased the survival rate of myocardial cells. CUEDC2was involved in cardiac myocytesdeath by interacting with ASK1and promoting JNK sustained activation. Interestingly,there was no significant difference in the activity of NF-κB between WT and Cuedc2^-/-miceunder ischemia-reperfusion injury.3. Cardiac hypertrophy and heart failure mouse model were established in the way ofleft ventricular long-term overload stress induced by aortic coarctation (TAC). CUEDC2protein expression in myocardial tissue decreased after TAC2weeks; knockout ofCUEDC2gene could not affect the degree of cardiac hypertrophy, but significantlyinhibited the development of cardiac insufficiency; two weeks after TAC, with isolatingmouse cardiomyocytes, and measuring single cell contractility and calcium handlingcapacity, we found that myocardial contraction force and calcium handling capacity inCuedc2^-/-cell was better than the wild-type cardiomyocytes; four weeks after the TAC, withtotal protein extraction of myocardial tissue to detect the expression of SERCA2a protein,we found SERCA2a protein level was higher in Cuedc2^-/-mice, indicating that the increasein SERCA2a stability; CUEDC2may participate in the occurance of ventricular overload-induced cardiac dysfunction by affecting the stability of SERCA2a protein andactivity.4. In myocardial tissue from cyanotic patients, CUEDC2protein expression wassignificantly decreased. In hypoxic cultured cardiac myocytes, CUEDC2protein expressionlevel also decreased significantly, indicating that the gene CUEDC2may also play animportant role during long time ER stress. In the chronic hypoxic cardiac tissue, we foundthat only ATF6α branch activity enhanced by detecting activity of the UPR three branch,and that IREα and PERK signaling pathway activity had no significant change; we furtherlyidentified the increase of ATF6α activity in the long-time hypoxic cultured cardiacmyocytes; knockdown the expression of ATF6α could lead to the increase of myocardialcell apoptosis during prolonged hypoxia and hypoxia reoxygenation, showing that theATF6α activation is an important regulatory factor of chronic myocardial hypoxiaadaptation.Conclusions:1. CUEDC2highly expressed in the heart, but CUEDC2gene knockout does not affectthe growth, development and physiological heart function in mice.2. CUEDC2gene knockout could significantly reduce myocardial cell death duringischemia-reperfusion injury, also the myocardial infarct size. CUEDC2interactes withASK1and promotes JNK sustained activation, leading to cell death.3. CUEDC2gene knockout could not affect the ventricular long overload-inducedcardiac hypertrophy, but can significantly inhibit the development of cardiac insufficiency.Regulating SERCA2a protein stability and activity is the molecular mechanism in whichCUEDC2participates in the development and progression of cardiac dysfunction. CUEDC2may play a negative role in myocardial cell function during chronic ER stress.4. CUEDC2protein expression was decreased during chronic hypoxia, also the activityof ATF6α. ATF6α plays an important role in myocaral adaptation to chronic hypoxia, andprotects cardiac myocytes under hypoxia-reoxygenation injury.